Abstract
Previous studies have reported that microRNA-30e (miR-30e) is dysregulated in multiple human cancers. However, the expression, functions and molecular mechanism of miR-30e in NSCLC remain unknown. In this study, we found that miR-30e was expressed at a low level in NSCLC tissues and cell lines. In NSCLC cell lines, enforced expression of miR-30e could inhibit cell proliferation and invasion in vitro. In addition, miR-30e negatively regulated SOX9 expression through directly binding to the 3′UTR of SOX9, and an inverse correlation was found between miR-30e and SOX9 mRNA expression in NSCLC tissues. Moreover, knockdown of SOX9 led to decreased proliferation and invasion of NSCLC cells. Taken together, miR-30e acts as a tumor suppressor in NSCLC, and inhibits cell proliferation and invasion possibly by directly targeting SOX9. These findings might provide novel therapeutic targets for NSCLC.
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Introduction
Lung cancer is one of the most common types of malignancy in humans and a leading cause of cancer-related death among both men and women around the world [1, 2]. Non-small cell lung cancer(NSCLC), an aggressive type of lung cancer, has a higher tumor recurrence and metastasis [3, 4]. The 5-year survival rate of NSCLC patients still has shown no significant increase [2], Therefore, development of novel effective therapeutic strategies for patients with NSCLC is essential.
MicroRNAs (miRNAs) are a new series of endogenous, single-stranded and non-coding RNAs, whose length is about 21–25 nucleotides [5]. miRNAs can directly bind to the 3′-untranslated region (3′UTR) of their target genes to promote gene degradation or inhibit translation [6, 7]. miRNAs have been demonstrated to be involved in cell proliferation, differentiation, migration, invasion and other biological processes [8,9,10]. Especially, abnormally expressed miRNAs have been observed in a wide range of human cancer. Moreover, miRNAs may function either as oncogenes or tumor suppressor, depending on the regulated tumor types and their targeted genes [11, 12]. These findings suggested that miRNAs might be a therapeutic target for human cancers.
In this study, we found that miR-30e was decreased in NSCLC tissues and cell lines by qRT-PCR. Function analysis showed that miR-30e overexpression suppressed NSCLC cell proliferation and invasion in vitro. We further identified SOX9 as a target for miR-30e.Taken together, our data suggested that miR-30e could inhibit NSCLC progression via SOX9.
Materials and methods
Human tissues
NSCLC tissues and paired normal adjacent tissues (NATs) were obtained from 52 patients undergoing surgery resection at the Department of Thoracic surgery (The First Affiliated Hospital of Dalian Medical University, China). None of these patients received adjuvant therapy before surgery. Both NSCLC tissues and NATs were immediately frozen in liquid nitrogen and then stored at − 80 °C prior to RNA isolation. Written informed consent was provided by all patients and this study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Dalian Medical University.
Cell lines and cell culture
Four NSCLC cell lines (H1299, H460, SPC-A1, A549) and one normal human bronchial epithelial cell line (16HBE) were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY) at 37 °C in a humidified atmosphere with 5% CO2.
Cell transfection
miR-30e mimics, miRNA mimic negative control (miR-NC), small interfering RNA(siRNA) targeting SOX9 (si-SOX9) and its negative control (si-NC) were generated by GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was adopted to perform cell transfection following the manufacturer’s protocol.
Quantitative real-time PCR
Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified using an ND-2000 spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific, Inc., Wilmington, DE, USA), following the manufacturer’s protocols. Reverse transcription was carried out with M-MLV (Promega, Madison, WI, USA). Relative expressions of miR-30e and SOX9 mRNA were detected by SYBR Premix Ex Taq™Kits (TaKaRa, Tokyo, Japan) using Applied Biosystems® 7900HT Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). U6 (for miRNAs) and GAPDH (for SOX9) were used as control. Relative expression levels were determined according to the 2-∆∆Ct method. Primer: miR-30e forward 5′-GTGCCTCACTGCGTCTC-3′, reverse 5′-GAAAGCCGGTGCGTAGCTG-3′; SOX9 forward 5′-AGGAAGTCGGTGAAGA ACGG-3′, reverse 5′-CGCCTTGAAGATGGCGTTG-3′; GAPDH forward 5′-TGACTTCAA CAGCGACACCCA-3′, reverse 5′-CACCCTGTTGCTGTAGCCAAA-3′; U6 forward 5′-GACCGAGTGTAGCAAGG-3′, reverse 5′-GTTCTTCCGAGAACATATAC-3′.
MTT assay
Transfected cells were harvested 24 h after transfection and re-seeded in 96- well plates at a density of 3000 cells/well. Cells were then incubated at 37 °C for 24, 48, 72, and 96 h. At each time point, 20µL MTT solution (5 mg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added in each well and incubated for another 4 h at 37 °C. The culture medium was removed and 150µL dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) was added into each well. Finally, the optical density (OD) at 490 nm was detected using an enzyme-linked immunosorbent assay reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Cell invasion assay
Cell invasion ability was detected using transwell chambers (8 µm pore size; BD Biosciences, San Jose, CA, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). Transfected cells were harvested 24 h after transfection, and 5 × 104 transfected cells were washed twice with PBS, resuspended in 200 µL serum-free DMEM and added into the upper chamber. 500 µL DMEM containing 20% fetal bovine serum (FBS) was filled into the lower chamber as a chemoattractant. After incubation at 37 °C for 48 h, non-invaded cells were wiped away carefully with cotton wool. The invaded cells on the lower surface were fixed in 4% paraformaldehyde, stained with 0.5% crystal violet, photographed and counted in five randomly selected areas under a 200× microscope field.
Bioinformatics prediction and luciferase reporter assay
TargetScan (http://www.targetscan.org/) and miRanda (http://www.Microrna.org/microrna/) were used to predict the potential targets of miR-30e.
For luciferase reporter assay, HEK293T cells (Shanghai Institute of Biochemistry and Cell Biology, China) were co-transfected with pmirGLO-SOX9-3′UTR wild-type (Wt) or pmirGLO-SOX9-3′UTR mutant (Mut), and miR-30e mimics or miR-NC using Lipofectamine 2000 according to the manufacturer’s instructions. After incubation for 48 h at 37 °C, cells were harvested and luciferase activity was detected using Dual-Luciferase® Reporter Assay system (Promega Corporation, Madison, WI, USA) following the manufacturer’s protocol.
Western blot and antibody
The cells were harvested and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany). The protein concentration was measured using the BCA assay (KeyGen Biotech). Equal amounts of protein were separated by 10% SDS–polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with the specific antibodies (appropriate concentration referred to specification) and the protein band was detected with enhanced chemiluminescence system.
Antibody: SOX9 antibody (1:1000 dilution; sc-166505; Santa Cruz Biotechnology, CA, USA), GADPH antibody (1:1000 dilution; sc-166574; Santa Cruz Biotechnology, CA, USA), goat anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (1:5000 dilution; Abcam, Cambridge, UK).
Statistical analysis
All data were presented as the mean ± standard deviation. SPSS 15.0 was used for statistical analyses. P < 0.05 was considered as statistically significant difference.
Results
MiR-30e is downregulated in NSCLC
The expressions of miR-30e in 52 pairs of NSCLC tissues and matched NATs were detected by using qRT-PCR. The results showed that miR-30e was significantly downregulated in 52 NSCLC tissues compared with NATs (Fig. 1a, P < 0.05). Then, the expression level of miR-30e was detected in four NSCLC cell lines and one normal human bronchial epithelial cell line (16HBE). As shown in Fig. 1b, there was lower expression of miR-30e in NSCLC cell lines than in HBE (P < 0.05). These results suggested that lower level miR-30e may be associated with NSCLC carcinogenesis and progression, and should be considered a potential biomarker for predicting poor prognosis in NSCLC.
MiR-30e overexpression suppresses NSCLC cell proliferation and invasion in vitro
To investigate the effect of miR-30e overexpression on cell proliferation and invasion of NSCLC, H1299 and A549 cells were transfected with miR-30e mimics or miR-NC. After 48 h transfection, miR-30e was significantly increased in H1299 and A549 cells transfected with miR-30e mimics compared with miR-NC (Fig. 2a, P < 0.05). It showed that miR-30e mimics were effective as endogenous miR-30e in vitro. In addition, following transfection, MTT assay and cell invasion assay were performed. As shown in Fig. 2b, c, upregulation of miR-30e obviously suppressed the proliferation (P < 0.05) and invasion (P < 0.05) of H1299 and A549 cells.
SOX9 is the direct target gene of miR-30e
To explore the underlying mechanism of miR-30e in NSCLC, bioinformatics analysis was performed to predict the targets of miR-30e. SOX9 was identified as a candidate target of miR-30e (Fig. 3a,). qRT-PCR and western blot revealed that SOX9 expression was downregulated in miR-30e mimics-transfected H1299 and A549 cells at both mRNA and protein level as predicted (Fig. 3b, c, P < 0.05). To examine whether the 3′UTR of SOX9 could be directly targeted by miR-30e, luciferase reporter assay was performed. The result showed (Fig. 3d) that restoring the expression of miR-30e decreased the luciferase activities of pmirGLO-SOX9-3′UTR Wt (P < 0.05), but had no effect on pmirGLO-SOX9-3′UTR Mut. In addition, SOX9 expression in 52 pairs of NSCLC tissues and NATs was measured using qRT-PCR. SOX9 mRNA expression levels were evaluated in NSCLC tissues compared with NATs as expected (Fig. 3e, P < 0.05). Moreover, a statistically significant inverse correlation was observed by Spearman’s correlation analysis between miR-30e and SOX9 mRNA expression level in NSCLC tissues (Fig. 3f, r = − 0.5626, P < 0.001). Collectively, these results suggested that SOX9 is a direct target of miR-30e.
Downregulation of SOX9 inhibited H1299 and A549 cell proliferation and invasion in vitro
To confirm whether the tumor suppressor role of miR-30e in NSCLC is mediated by SOX9, we knocked down SOX9 in H1299 and A549 cells. Western blot analysis showed that SOX9 was downregulated in H1299 and A549 cells, following transfection with si-SOX9 (Fig. 4a, P < 0.05). Following transfection, MTT assay and cell invasion assay showed that si-SOX9 obviously inhibited cell proliferation and invasion of H1299 and A549 cells (Fig. 4b, c, P < 0.05). These results further suggested that SOX9 was the direct functional target of miR-30e in NSCLC.
Discussion
NSCLC is the most common type of lung cancer and has the highest mortality rate in China [13]. However, the mechanisms underlying NSCLC occurrence and development have largely remained elusive. Therefore, it is urgent to explore novel therapeutic targets for NSCLC patients. miRNA-based anti-tumor treatments are being developed to target multiple cancer-driven effector genes in tumor-suppressing and oncogenic signaling pathways involved in cell proliferation, apoptosis, invasion, metastasis, and survival [14].
Previous studies have reported that miR-30e was abnormal in multiple types of human cancer. miR-30e functions as a tumor suppressor in human malignancies by affecting different signaling pathways [15, 16, 17]. These findings indicated that miR-30e could be a diagnostic, prognostic biomarker and therapeutic target for human cancers. Here, we found that miR-30e is significantly downregulated in NSCLC tissues and NSCLC cell lines. In addition, overexpression of miR-30e suppressed cell proliferation and invasion of NSCLC. These results suggested that miR-30e played a crucial role in NSCLC growth and metastasis.
Reports have showed that miRNAs need to act on the 3′UTR of target genes. The identification of miR30e targets is important for understanding its roles in tumorigenesis and tumor development. To explore the molecular mechanism underlying the tumor-suppressive roles of miR-30e in NSCLC, bioinformatics analysis was performed to predict targets for miR-30e.In multiple potential target genes, SOX9 was selected for further validation since SOX9 has been demonstrated to be involved in NSCLC carcinogenesis and progression [18]. In addition, SOX9 was identified as a direct target of many diverse miRNAs in NSCLC, including miR-124 [19], miR-206 [20], and miR-32 [21]. Here, we further confirmed that SOX9 was a direct and functional target gene of miR-30e by using luciferase reporter assay, qRT-PCR and western blot analysis. Furthermore, SOX9 mRNA was increased and inversely correlated with miR-30e expression level in NSCLC tissues. Moreover, SOX9 downregulation had similar effects with miR-30e overexpression on proliferation and invasion in NSCLC cells. Taken together, our current study verified that the biological roles of miR-30e on NSCLC were possibly via negative regulation of the expression of its novel identified target, SOX9.
SOX9, a member of the sex-determining region Y (SRY) box family, has been demonstrated to be frequently upregulated in various human cancers such as prostate cancer [22], breast cancer [23] and colorectal cancer [24]. A study by Jiang et al. showed that SOX9 was expressed in high levels in lung adenocarcinoma. Lower expression of SOX9 reduced adhesive and anchorage-independent growth and decreased tumorigenicity in vivo [25]. Zhou et al. reported that SOX9 mRNA and protein were particularly overexpressed in NSCLC tissues and cell lines and obviously associated with the histological stage of NSCLC patients. Multivariate analysis illustrated that SOX9 high expression may be an independent prognostic indicator for the survival of NSCLC patients [26]. Functional experiments indicated that SOX9 knockdown suppressed NSCLC cells growth and invasion [3]. These findings suggested that miR-30e/SOX9-based targeted therapy could be investigated as a new therapeutic strategy for NSCLC.
In summary, this study showed that miR-30e was downregulated in both NSCLC tissues and cell lines. We found that the expression of miR-30e in H1299 and A549 cells could inhibit proliferation and invasion, potentially by directly targeting SOX9. These findings of this study have provided novel therapeutic targets for the future treatments of patients with NSCLC. In the future, we will further explore the differences in the expression of these two genes in different types of lung cancer, and also further study the clinical significance of these two genes that provide a better diagnosis and treatment basis in lung cancer.
References
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108.
Fiteni F, Anota A, Westeel V, Bonnetain F. Methodology of health-related quality of life analysis in phaseIII advanced non-small-cell lung cancer clinical trials: a critical review. BMC Cancer. 2016;16:122.
Li Z, Song Y, Liu L, et al. miR-199a impairs autophagy and induces cardiac hypertrophy through mTOR activation. Cell Death Differ. 2017;24(7):1205–13.
Mao M, Wu Z, Chen J. MicroRNA-187-5p suppresses cancer cell progression in non small cell lung cancer(NSCLC) through down-regulation of CYP1B1. Biochem Biophys Res Commun. 2016;478:649–55.
Kim VN. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol. 2005;6:376–85.
Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer. 2006;6:857–66.
Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33.
Aigner A. MicroRNAs (miRNAs) in cancer invasion and metastasis: therapeutic approaches based on metastasis-related miRNAs. J Mol Med (Berl). 2011;89:445–57.
Rottiers V, Naar AM. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol. 2012;13:239–50.
Cho WC. MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy. Int J Biochem Cell Biol. 2010;42:1273–81.
Xia H, Li Y, Lv X. MicroRNA-107 inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer. Int J Oncol. 2016;49(4):1325–33.
Lu C, Xie Z, Peng Q. MiRNA-107 enhances chemosensitivity to paclitaxel by targeting antiapoptotic factor Bcl-w in non small cell lung cancer. Am J Cancer Res 2017,7(9):1863.
Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66:115–32.
Garzon R, Marcucci G, Croce CM. Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov. 2010;9:775–89.
Lin Z, Li JW, Wang Y, et al. Abnormal miRNA-30e expression is associated with breast cancer progression. Clin Lab. 2016;62:121–8.
Kwak S, Kim B, Ahn H, et al. Ionizing radiation-inducible miR-30e promotes glioma cell invasion through EGFR stabilization by directly targeting CBL-B. Febs J. 2015;282(8):1512–25.
Hershkovitzrokah O, Modai S, Pasmanikchor M, et al. MiR-30e induces apoptosis and sensitizes K562 cells to imatinib treatment via regulation of the BCR-ABL protein. Cancer Lett. 2015;356(2):597–605.
Wang X, Ju Y, Zhou MI, Liu X, Zhou C. Upregulation of SOX9 promotes cell proliferation, migration and invasion in lung adenocarcinoma. Oncol Lett. 2015;10:990–4.
Wang X, Liu Y, Liu X, Yang J, Teng G, Zhang L, Zhou C. MiR-124 inhibits cell proliferation, migration and invasion by directly targeting SOX9 in lung adenocarcinoma. Oncol Rep. 2016;35:3115–21.
Zhang YJ, Xu F, Zhang YJ, Li HB, Han JC, Li L. miR-206 inhibits non small cell lung cancer cell proliferation and invasion by targeting SOX9. Int J Clin Exp Med. 2015;8:9107–13.
Zhu D, Chen H, Yang X, Chen W, Wang L, Xu J, Yu L. miR-32 functions as a tumor suppressor and directly targets SOX9 in human non-small cell lung cancer. Onco Targets Ther. 2015;8:1773–83.
Wang H, Leav I, Ibaragi S, Wegner M, Hu GF, Lu ML, Balk SP, Yuan X. SOX9 is expressed in human fetal prostate epithelium and enhances prostate cancer invasion. Cancer Res. 2008;68:1625–30.
Muller P, Crofts JD, Newman BS, Bridgewater LC, Lin CY, Gustafsson JA, Strom A. SOX9 mediates the retinoic acid-induced HES-1 gene expression in human breast cancer cells. Breast Cancer Res Treat. 2010;120:317–26.
Bruun J, Kolberg M, Nesland JM, Svindland A, Nesbakken A, Lothe RA. Prognostic significance of beta-catenin, e-cadherin, and SOX9 in colorectal cancer: results from a large population-representative series. Front Oncol. 2014;4:118.
Jiang SS, Fang WT, Hou YH, Huang SF, Yen BL, Chang JL, Li SM, Liu HP, Liu YL, Huang CT, Li YW, Jang TH, Chan SH, Yang SJ, Hsiung CA, Wu CW, Wang LH. Chang IS. Upregulation of SOX9 in lung adenocarcinoma and its involvement in the regulation of cell growth and tumorigenicity. Clin Cancer Res. 2010;16:4363–73.
Zhou CH, Ye LP, Ye SX, Li Y, Zhang XY, Xu XY, Gong LY. Clinical significance of SOX9 in human non-small cell lung cancer progression and overall patient survival. J Exp Clin Cancer Res. 2012;31:18.
Acknowledgements
This work was supported by a grant from the National Nature Science Foundation of China (181774078, 81773453, 81803886), National Nature Science Foundation of Liaoning Province, China (201602227, 20170540300).
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Cui, Y., Zhao, L., Zhao, S. et al. MicroRNA-30e inhibits proliferation and invasion of non-small cell lung cancer via targeting SOX9. Human Cell 32, 326–333 (2019). https://doi.org/10.1007/s13577-018-0223-0
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DOI: https://doi.org/10.1007/s13577-018-0223-0